Compositions that preferentially potentiate subtypes of GABAA receptors and methods of use thereof

ABSTRACT

The invention provides compositions containing isomerically pure forms of neurosteroids that permit preferential modulation of different subtypes of GABAA receptors, such as preferential modulation of α4β3δ GABAA receptors over α1β2γ2 GABAA receptors. The invention also provides methods of treating GABAA disorders using such compositions.

This application claims the benefit of, and priority to, U.S.Non-provisional application Ser. No. 16/587,157, filed Sep. 30, 2019,the contents of which are incorporated by reference.

FIELD OF THE INVENTION

The invention relates generally to compositions containing neurosteroidsand methods of using the same to treat GABA_(A) disorders in a subject.

BACKGROUND

According the World Health Organization (WHO), neurological disordersaffect up to one billion people worldwide. Neurological disordersinclude a wide range of conditions, such as Alzheimer's disease, braininjuries, epilepsy, headache, infections, multiple sclerosis, andParkinson's disease, and stroke. Many neurological disorders stem fromaltered signaling by receptors for the neurotransmitter γ-aminobutyricacid (GABA). GABA_(A) receptors are pentameric transmembrane receptorsthat include various combinations of 19 different subunit polypeptides.At least 15 GABA_(A) receptor subtypes are known, and particularsubtypes are associated with different conditions. For example, alteredactivity of receptor subtypes that include α₂ or α₃ subunits isassociated with anxiety disorders, whereas α₅-containing subtypes appearto play a role in memory and cognition.

Neuroactive steroids that alter the activity of GABA_(A) receptors havebeen investigated as drug candidates for a variety of neurologicaldisorders. However, the therapeutic potential of such molecules remainslargely untapped. One reason for the shortfall is that the large numberof chemical variants that can be made from the steroid structural coremakes it difficult to know whether compounds currently beinginvestigated have superior pharmacological properties to other moleculesthat have not yet been made or analyzed. Another issue is that thestructural similarity of different GABA_(A) receptor subtypes makes itchallenging to identify molecules with a desired subtype specificity.Consequently, millions of people continue to suffer from neurologicalconditions due to the limited arsenal of neuroactive steroids currentlyat our disposal.

SUMMARY

The invention provides compositions that contain isomerically pure formsof selected neurosteroids. The invention recognizes that interactionsbetween neurosteroids and GABA_(A) receptors are highly sensitive to thestereochemical structure of the neurosteroids and that certainneurosteroids display strong GABA_(A) receptor subtype specificity whenprovided in compositions that are substantially free of isomericcontaminants. Because the compositions of the invention selectivelytarget particular GABA_(A) receptor subtypes, they have greatly improvedpharmacological efficacy over prior compositions, including those thatcontain biologically active compounds contaminated with isomers that areless active.

In a particular embodiment, the invention provides compositions thatcontain an isomerically pure form of a compound of Formula (I):

The present invention includes the finding that isomerically purecompositions of Formula (I) are considerably more active on α4β3δGABA_(A) receptors than on α1β2γ2 GABA_(A) receptors. As describedherein, such compositions modulate the activity of α4β3δ receptors withan EC₅₀ at least 5-fold lower than for α1β2γ2 receptors. Without wishingto be bound by a particular theory, it is believed that thestereochemical configurations at all of the chiral centers of themolecule and the atomic bonding patterns within the molecule areimportant in conferring GABA_(A) receptor subtype selectivity. Thus,mixtures that contain the compound of Formula (I) together with isomersthereof, such as regioisomers of Formula (I) or stereoisomers thatdiffer structurally from Formula (I) at only a single chiral center,lack such selectivity. Because compositions that contain an isomericallypure form of the compound of Formula (I) preferentially target α4β3δGABA_(A) receptors, they are useful for therapeutic applications inwhich altering the activity of this receptor is beneficial.

In an aspect, the invention provides pharmaceutical compositionscontaining an isomerically pure form of a compound of Formula (I):

wherein the compound of Formula (I) is present in a therapeuticallyeffective amount to preferentially potentiate an α4β3δ GABA_(A) receptoras compared to an α1β2γ2 GABA_(A) receptor.

In another aspect, the invention provides methods for treating aGABA_(A) disorder by providing to a subject a pharmaceutical compositioncomprising an isomerically pure form of a compound of Formula (I):

wherein the compound of Formula (I) is present in a therapeuticallyeffective amount to preferentially potentiate an α4β3δ GABA_(A) receptoras compared to an α1β2γ2 GABA_(A) receptor.

The composition may be chemically pure, i.e., free from other moleculesor chemical species. For example, the other molecule or chemical speciesmay have a distinct chemical formula, structural formula, empiricalformula, molecular formula, or condensed formula. The composition mayhave a defined level of chemical purity. For example, the compound ofFormula (I) may be present at at least 95% by weight, at least 96% byweight, at least 97% by weight, at least 98% by weight, at least 99% byweight, at least 99.5% by weight, at least 99.6% by weight, at least99.7% by weight, at least 99.8% by weight, or at least 99.9% by weightof the total amount of a mixture that includes the compound of Formula(I) and one or more distinct molecules or chemical species.

The composition may be isomerically pure with respect to all isomers.The composition may be isomerically pure with respect to one or moreparticular types of isomers. The composition may be substantially freeof structural isomers or a particular type of structural isomers, suchas a regioisomers. The composition may be substantially free ofstereoisomers or a particular type of stereoisomers, such as enantiomersor diastereomers.

The composition may contain the compound of Formula (I) at any level ofisomeric purity to achieve preferential modulation of an α4β3δ GABA_(A)receptor as compared to an α1β2γ2 GABA_(A) receptor. For example, thecompound of Formula (I) may be present at at least 95% by weight, atleast 96% by weight, at least 97% by weight, at least 98% by weight, atleast 99% by weight, at least 99.5% by weight, at least 99.6% by weight,at least 99.7% by weight, at least 99.8% by weight, or at least 99.9% byweight of the total amount of isomeric molecules that include thecompound of Formula (I) and an isomer thereof.

The composition may contain the compound of Formula (I) and besubstantially free of stereoisomers. The stereoisomer may differ fromFormula (I) at one, two, three, four, five, six, seven, or eight chiralcenters. The stereoisomer may be a diastereomer or an enantiomer. Forexample, the stereoisomer may be a compound of Formulas (II) or (III):

The composition may contain one or more stereoisomers of the compound ofFormula (I), such as a compound of Formula (II) or (III), at less than5%, less than 4%, less than 3%, less than 2%, less than 1%, less than0.5%, or less than 0.1% of the total of the compound of Formula (I) andthe one or more stereoisomers thereof. The composition may contain thecompound of Formula (I) and one or more stereoisomer thereof at a ratioof at least 19:1, 20:1, 25:1, 30:1, 40:1, 50:1, 100:1, 200:1, 500:1, or1000:1.

The compound may potentiate a GABA_(A) receptor, a GABA_(A) receptorsubtypes, or a subset of GABA_(A) receptor subtypes by any mechanism.The compound may potentiate a GABA_(A) receptor, subtype, or subset byallosteric modulation, activation, or inhibition. The allostericmodulation may be positive or negative.

The composition may preferentially potentiate an α4β3δ GABA_(A) receptoras compared to an α1β2 γ2 GABA_(A) receptor to any degree. Thecomposition may preferentially potentiate an α4β3δ GABA_(A) receptor ascompared to an α1β2γ2 GABA_(A) receptor by any measure or parameter.

The composition may have an EC₅₀ for α4β3δ GABA_(A) receptors that islower than the EC₅₀ for α1β2γ2 GABA_(A) receptors. The EC₅₀ for α4β3δGABA_(A) receptors may be lower than the EC₅₀ for α1β2γ2 GABA_(A)receptors by about 2-fold, about 3-fold, about 4-fold, about 5-fold,about 6-fold, about 7-fold, about 8-fold, about 10-fold, about 20-fold,about 50-fold, about 100-fold, about 200-fold, about 500-fold, or about1000-fold. The EC₅₀ for α4β3δ GABA_(A) receptors may be less than about50%, less than about 40%, less than about 30%, less than about 25%, lessthan about 20%, less than about 15%, less than about 10%, less thanabout 5%, less than about 4%, less than about 3%, less than about 2%,less than about 1%, less than about 0.5%, less than about 0.2%, or lessthan about 0.1% of the EC₅₀ for α1β2γ2 GABA_(A) receptors.

The composition may have a binding affinity (which may be expressed,e.g., as a dissociation constant K_(D)) for α4β3δ GABA_(A) receptorsthat is lower than the binding affinity for α1β2γ2 GABA_(A) receptors.The binding affinity for α4β3δ GABA_(A) receptors may be lower than thebinding affinity for α1β2γ2 GABA_(A) receptors by about 2-fold, about3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about8-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold,about 200-fold, about 500-fold, or about 1000-fold. The binding affinityfor α4β3δ GABA_(A) receptors may be less than about 50%, less than about40%, less than about 30%, less than about 25%, less than about 20%, lessthan about 15%, less than about 10%, less than about 5%, less than about4%, less than about 3%, less than about 2%, less than about 1%, lessthan about 0.5%, less than about 0.2%, or less than about 0.1% of thebinding affinity for α1β2γ2 GABA_(A) receptors.

The composition may have an EC₅₀ for α4β3δ GABA_(A) receptors that isbelow a defined value. The composition may have an EC₅₀ for α4β3δGABA_(A) receptors that is less than about 1 μM, less than about 500 nM,less than about 400 nM, less than about 300 nM, less than about 200 nM,less than about 100 nM, less than about 50 nM, less than about 25 nM,less than about 10 nM, less than about 5 nM, less than about 2.5 nM,less than about 1 nM, less than about 0.5 nM, less than about 0.25 nM,or less than about 0.1 nM.

The composition may have a binding affinity for α4β3δ GABA_(A) receptorsthat is below a defined value. The composition may have an bindingaffinity for α4β3δ GABA_(A) receptors that is less than about 1 μM, lessthan about 500 nM, less than about 400 nM, less than about 300 nM, lessthan about 200 nM, less than about 100 nM, less than about 50 nM, lessthan about 25 nM, less than about 10 nM, less than about 5 nM, less thanabout 2.5 nM, less than about 1 nM, less than about 0.5 nM, less thanabout 0.25 nM, or less than about 0.1 nM.

The composition may be effective for treatment of a GABA_(A) disorder.The GABA_(A) disorder may be any disease, disorder, or conditionassociated with altered GABA_(A) receptor function or any disorder maybe disease, disorder, or condition that can be ameliorated by alteredGABA_(A) receptor function. The GABA_(A) disorder may be acute pain, anaddictive disorder. Alzheimer's disease, Angelman's syndrome,anti-social personality disorder, an anxiety disorder, attention deficithyperactivity disorder (ADHD), an attention disorder, an auditorydisorder, autism, an autism spectrum disorder, bipolar disorder, chronicpain, a cognitive disorder, a compulsive disorder, a convulsivedisorder, dementia, depression, dysthymia, an epileptic disorder,essential tremor, epileptogenesis, fragile X syndrome, generalizedanxiety disorder (GAD), Huntington's disease, injury related painsyndrome, insomnia, ischemia, Lewis body type dementia, a memorydisorder, migraines, a mood disorder, movement disorder, aneurodegenerative disease, neuropathic pain, an obsessive compulsivedisorder, pain, a panic disorder, Parkinson's disease, a personalitydisorder, posttraumatic stress disorder (PTSD), psychosis, Rettsyndrome, a schizoaffective disorder, schizophrenia, a schizophreniaspectrum disorder, a seizure disorder, a sleep disorder, social anxietydisorder, status epilepticus, stress, stroke, tinnitus, traumatic braininjury (TBI), vascular disease, vascular malformation, vascular typedementia movement disorder, Wilson's disease, or withdrawal syndrome.

The composition may be formulated for administration by a particularmechanism. The composition may be formulated for oral, intravenous,enteral, parenteral, dermal, buccal, topical nasal, or pulmonaryadministration. The composition may be formulated for administration byinjection or on an implantable medical device (e.g., stent ordrug-eluting stent or balloon equivalents).

The composition may be formulated a single daily dosage. The compositionmay be formulated for multiple daily dosages, e.g., two, three, four,five, six or more daily dosages.

The composition may be provided to the subject according to any dosingschedule. The composition may be provided once per day. The compositionmay be provided multiple times per day. The composition may be providedtwo time, three times, four times, five times, six times, or more perday.

DETAILED DESCRIPTION

The invention provides compositions that contain isomerically pure formsof neurosteroids and methods of using such compositions to treatneurological and other disorders. The invention is based on therecognition that isomerically pure neurosteroids allow modulation ofspecific subtypes of γ-aminobutyric acid (GABA) receptors. Because thecompositions permit selective modulation of subtypes of GABA receptors,they are useful for treating conditions in which alteration of thosereceptor subtypes provides therapeutic benefit.

Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75th Ed., inside cover, and specificfunctional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5th Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3rd Edition, CambridgeUniversity Press, Cambridge, 1987.

As used herein, a “pure isomeric” compound or “isomerically pure”compound is substantially free of other isomers of the compound. Theterm “pure isomeric” compound or “isomerically pure” denotes that thecompound comprises at least 95% by weight, at least 96% by weight, atleast 97% by weight, at least 98% by weight, at least 99% by weight, atleast 99.5% by weight, at least 99.6% by weight, at least 99.7% byweight, at least 99.8% by weight, or at least 99.9% by weight of thecompound with the specified structure. In certain embodiments, theweights are based upon total weight of all isomers of the compound.

As used herein, a “pure stereoisomeric” compound or “stereoisomericallypure” compound is substantially free of other stereoisomers of thecompound. Thus, the composition is substantially free of isomers thatdiffer at any chiral center. If the compound has multiple chiralcenters, a substantial majority of the composition contains compoundshaving identical stereochemistry at all of the chiral centers. The term“pure stereoisomeric” compound or “stereoisomerically pure” denotes thatthe compound comprises at least 95% by weight, at least 96% by weight,at least 97% by weight, at least 98% by weight, at least 99% by weight,at least 99.5% by weight, at least 99.6% by weight, at least 99.7% byweight, at least 99.8% by weight, or at least 99.9% by weight of thecompound with the specified stereochemistry. In certain embodiments, theweights are based upon total weight of all stereoisomers of thecompound.

As used herein, a pure enantiomeric compound is substantially free fromother enantiomers or stereoisomers of the compound (i.e., inenantiomeric excess). In other words, an “S” form of the compound issubstantially free from the “R” form of the compound and is, thus, inenantiomeric excess of the “R” form. The term “enantiomerically pure” or“pure enantiomer” denotes that the compound comprises at least 95% byweight, at least 96% by weight, at least 97% by weight, at least 98% byweight, at least 99% by weight, at least 99.5% by weight, at least 99.6%by weight, at least 99.7% by weight, at least 99.8% by weight, or atleast 99.9% by weight of the enantiomer. In certain embodiments, theweights are based upon total weight of all enantiomers or stereoisomersof the compound.

Compounds described herein may also comprise one or more isotopicsubstitutions. For example, H may be in any isotopic form, including ¹H,²H (D or deuterium), and ³H (T or tritium); C may be in any isotopicform, including ¹²C, ¹³C, and ¹⁴C; N may be any isotopic form, including¹⁴N and ¹⁵N; O may be in any isotopic form, including ¹⁶O and ¹⁸O; andthe like.

The articles “a” and “an” may be used herein to refer to one or to morethan one (i.e. at least one) of the grammatical objects of the article.By way of example “an analogue” means one analogue or more than oneanalogue.

As used herein, the terms “modulation” and “potentiation” refer to theinhibition or stimulation of GABA receptor function. A “modulator” or“potentiator” may be, for example, an agonist, partial agonist,antagonist, or partial antagonist of the GABA receptor. The “modulator”or “potentiator” may act at the active site or at an allosteric site ona GABA receptor. It may promote or inhibit ligand binding. It mayfacilitate or attenuate ligand-mediated, e.g., GABA-mediated, signaling.

“Pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound of theinvention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. In particular,such salts are non-toxic may be inorganic or organic acid addition saltsand base addition salts. Specifically, such salts include: (1) acidaddition salts, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or formed with organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N methylglucamine and thelike. Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe compound contains a basic functionality, salts of non-toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like. The term“pharmaceutically acceptable cation” refers to an acceptable cationiccounter-ion of an acidic functional group. Such cations are exemplifiedby sodium, potassium, calcium, magnesium, ammonium, tetraalkylammoniumcations, and the like. See, e.g., Berge, el al., J. Pharm. Sci. (1977)66(1): 1-79.

“Solvate” refers to forms of the compound that are associated with asolvent or water (also referred to as “hydrate”), usually by asolvolysis reaction. This physical association includes hydrogenbonding. Conventional solvents include water, ethanol, acetic acid, andthe like. The compounds of the invention may be prepared e.g. incrystalline form and may be solvated or hydrated. Suitable solvatesinclude pharmaceutically acceptable solvates, such as hydrates, andfurther include both stoichiometric solvates and non-stoichiometricsolvates. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and insoluble solvates. Representative solvates includehydrates, ethanolates and methanolates.

As used herein, the term “isotopic variant” refers to a compound thatcontains unnatural proportions of isotopes at one or more of the atomsthat constitute such compound. For example, an “isotopic variant” of acompound can contain one or more non-radioactive isotopes, such as forexample, deuterium (²H or D), carbon-13 (¹³C), nitrogen-15 (¹⁵N), or thelike. It will be understood that, in a compound where such isotopicsubstitution is made, the following atoms, where present, may vary, sothat for example, any hydrogen may be ²H/D, any carbon may be ¹³C, orany nitrogen may be ¹⁵N, and that the presence and placement of suchatoms may be determined within the skill of the art. Likewise, theinvention may include the preparation of isotopic variants withradioisotopes, in the instance for example, where the resultingcompounds may be used for drug and/or substrate tissue distributionstudies. The radioactive isotopes tritium, i.e., ³H, and carbon-14,i.e., ¹⁴C, are particularly useful for this purpose in view of theirease of incorporation and ready means of detection. Further, compoundsmay be prepared that are substituted with positron emitting isotopes,such as ¹¹C, ¹⁸F,¹⁵O, and ¹³N, and would be useful in Positron EmissionTopography (PET) studies for examining substrate receptor occupancy. Allisotopic variants of the compounds provided herein, radioactive or not,are intended to be encompassed within the scope of the invention.

“Stereoisomers”: It is also to be understood that compounds that havethe same molecular formula but differ in the nature or sequence ofbonding of their atoms or the arrangement of their atoms in space aretermed “isomers.” Isomers that differ in the arrangement of their atomsin space are termed “stereoisomers.” Stereoisomers that are not mirrorimages of one another are termed “diastereomers”, and those that arenon-superimposable mirror images of each other are termed “enantiomers.”When a compound has an asymmetric center, for example, and an atom, suchas a carbon atom, is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a “racemic mixture”.

“Tautomers” refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of n electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the aci- and nitro-forms of phenylnitromethane, that arelikewise formed by treatment with acid or base. Tautomeric forms may berelevant to the attainment of the optimal chemical reactivity andbiological activity of a compound of interest.

A “subject” to which administration is contemplated includes, but is notlimited to, a human (i.e., a male or female of any age group, e.g., apediatric subject (e.g., infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult or senior adult)) and/or anon-human animal, e.g., a mammal such as primates (e.g., cynomolgusmonkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents,cats, and/or dogs. In certain embodiments, the subject is a human. Incertain embodiments, the subject is a non-human animal.

Disease, disorder, and condition are used interchangeably herein.

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” contemplate an action that occurs while asubject is suffering from the specified disease, disorder or condition,which reduces the severity of the disease, disorder or condition, orretards or slows the progression of the disease, disorder or condition(“therapeutic treatment”), and also contemplates an action that occursbefore a subject begins to suffer from the specified disease, disorderor condition (“prophylactic treatment”).

In general, the “effective amount” of a compound refers to an amountsufficient to elicit the desired biological response, e.g., to treat aCNS-related disorder, is sufficient to induce anesthesia or sedation. Aswill be appreciated by those of ordinary skill in this art, theeffective amount of a compound of the invention may vary depending onsuch factors as the desired biological endpoint, the pharmacokinetics ofthe compound, the disease being treated, the mode of administration, andthe age, weight, health, and condition of the subject. An effectiveamount encompasses therapeutic and prophylactic treatment.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment of a disease, disorder orcondition, or to delay or minimize one or more symptoms associated withthe disease, disorder or condition. A therapeutically effective amountof a compound means an amount of therapeutic agent, alone or incombination with other therapies, which provides a therapeutic benefitin the treatment of the disease, disorder or condition. The term“therapeutically effective amount” can encompass an amount that improvesoverall therapy, reduces or avoids symptoms or causes of disease orcondition, or enhances the therapeutic efficacy of another therapeuticagent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to prevent adisease, disorder or condition, or one or more symptoms associated withthe disease, disorder or condition, or prevent its recurrence. Aprophylactically effective amount of a compound means an amount of atherapeutic agent, alone or in combination with other agents, whichprovides a prophylactic benefit in the prevention of the disease,disorder or condition. The term “prophylactically effective amount” canencompass an amount that improves overall prophylaxis or enhances theprophylactic efficacy of another prophylactic agent.

Compositions

Compounds

The invention provides compositions with isomerically pure forms ofneuro steroids.

In certain embodiments, the invention provides pharmaceuticalcompositions containing an isomerically pure form of a compound ofFormula (I):

The composition may be chemically pure, i.e., free from other moleculesor chemical species. For example, the other molecule or chemical speciesmay have a distinct chemical formula, structural formula, empiricalformula, molecular formula, or condensed formula. The composition mayhave a defined level of chemical purity. For example, the compound ofFormula (I) may be present at at least 95% by weight, at least 96% byweight, at least 97% by weight, at least 98% by weight, at least 99% byweight, at least 99.5% by weight, at least 99.6% by weight, at least99.7% by weight, at least 99.8% by weight, or at least 99.9% by weightof the total amount of a mixture that includes the compound of Formula(I) and one or more distinct molecules or chemical species.

The composition may contain the compound of Formula (I) at any level ofisomeric purity, i.e., the composition may contain the compound ofFormula (I) at a level in relation to an isomeric form of the compound.For example, the compound of Formula (I) may be present at at least 95%by weight, at least 96% by weight, at least 97% by weight, at least 98%by weight, at least 99% by weight, at least 99.5% by weight, at least99.6% by weight, at least 99.7% by weight, at least 99.8% by weight, orat least 99.9% by weight of the total amount of isomeric molecules thatinclude the compound of Formula (I) and an isomer thereof.

The composition may be isomerically pure with respect to all isomers.The composition may be isomerically pure with respect to one or moreparticular types of isomers. The composition may be substantially freeof structural isomers or a particular type of structural isomers, suchas a regioisomers. The composition may be substantially free ofstereoisomers or a particular type of stereoisomers, such as enantiomersor diastereomers.

The composition may contain the compound of Formula (I) at any level ofisomeric purity to achieve preferential modulation of an α4β3δ GABA_(A)receptor as compared to an α1β2γ2 GABA_(A) receptor. For example, thecompound of Formula (I) may be present at at least 95% by weight, atleast 96% by weight, at least 97% by weight, at least 98% by weight, atleast 99% by weight, at least 99.5% by weight, at least 99.6% by weight,at least 99.7% by weight, at least 99.8% by weight, or at least 99.9% byweight of the total amount of isomeric molecules that include thecompound of Formula (I) and an isomer thereof.

The composition may contain the compound of Formula (I) and besubstantially free of stereoisomers. The stereoisomer may differ fromFormula (I) at one, two, three, four, five, six, seven, or eight chiralcenters. The stereoisomer may be a diastereomer or an enantiomer. Forexample, the stereoisomer may be a compound of Formulas (II) or (III):

The composition may contain one or more stereoisomers of the compound ofFormula (I), such as a compound of Formula (II) or (III), at less than5%, less than 4%, less than 3%, less than 2%, less than 1%, less than0.5%, or less than 0.1% of the total of the compound of Formula (I) andthe one or more stereoisomers thereof. The composition may contain thecompound of Formula (I) and one or more stereoisomer thereof at a ratioof at least 19:1, 20:1, 25:1, 30:1, 40:1, 50:1, 100:1, 200:1, 500:1, or1000:1.

Formulations

The invention provides pharmaceutical compositions containing one ormore of the compounds described above. A pharmaceutical compositioncontaining the compounds may be in a form suitable for oral use, forexample, as tablets, troches, lozenges, fast-melts, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules, syrups or elixirs. Compositions intended for oral use may beprepared according to any method known in the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide pharmaceuticallyelegant and palatable preparations. Tablets contain the compounds inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example corn starch, or alginic acid; bindingagents, for example starch, gelatin or acacia, and lubricating agents,for example magnesium stearate, stearic acid or talc. The tablets may beuncoated or they may be coated by known techniques to delaydisintegration in the stomach and absorption lower down in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated by the techniques described in U.S. Pat. Nos. 4,256,108;4,166,452; and 4,265,874, the contents of which are incorporated hereinby reference, to form osmotic therapeutic tablets for control release.Preparation and administration of compounds is discussed in U.S. Pat.No. 6,214,841 and U.S. Pub. No. 2003/0232877, the contents of which areincorporated herein by reference.

Formulations for oral use may also be presented as hard gelatin capsulesin which the compounds are mixed with an inert solid diluent, forexample calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules in which the compounds are mixed with water or an oilmedium, for example peanut oil, liquid paraffin or olive oil.

An alternative oral formulation, where control of gastrointestinal tracthydrolysis of the compound is sought, can be achieved using acontrolled-release formulation, where a compound of the invention isencapsulated in an enteric coating.

Aqueous suspensions may contain the compounds in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents such as a naturally occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example, polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such a polyoxyethylene with partial esters derived from fattyacids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate. The aqueous suspensions may also contain one or morepreservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one ormore coloring agents, one or more flavoring agents, and one or moresweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the compounds in avegetable oil, for example, arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the compounds in admixturewith a dispersing or wetting agent, suspending agent and one or morepreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified, for example sweetening, flavoring and coloringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally occurring phosphatides, for example soya bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate and condensation products ofthe said partial esters with ethylene oxide, for example polyoxyethylenesorbitan monooleate. The emulsions may also contain sweetening andflavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, and agents for flavoringand/or coloring. The pharmaceutical compositions may be in the form of asterile injectable aqueous or oleaginous suspension. This suspension maybe formulated according to the known art using those suitable dispersingor wetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be in a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. In addition, fatty acidssuch as oleic acid find use in the preparation of injectables.

In certain embodiments, the formulation is not a sustained releaseformulation. In certain embodiments, the formulation is not injectable.In certain embodiments, the formulation does not contain particleshaving a D50 (volume weighted median diameter) of less than 10 microns.In certain embodiments, the formulation does not contain a polymersurface stabilizer. In certain embodiments, the formulation is not anaqueous suspension.

The composition may be formulated for administration by a particularmechanism. The composition may be formulated for oral, intravenous,enteral, parenteral, dermal, buccal, topical nasal, or pulmonaryadministration. The composition may be formulated for administration byinjection or on an implantable medical device (e.g., stent ordrug-eluting stent or balloon equivalents).

The composition may be formulated a single daily dosage. The compositionmay be formulated for multiple daily dosages, e.g., two, three, four,five, six or more daily dosages.

The composition may be provided to the subject according to any dosingschedule. The composition may be provide once per day. The compositionmay be provided multiple times per day. The composition may be providedtwo time, three times, four times, five times, six times, or more perday.

Treatment of GABA_(A) Receptor Disorders

The compositions of the invention are useful for treating disorders thatare associated with, or can be ameliorated by, alteration of activity ofa GABA_(A) receptor. GABA_(A) receptors are ligand-gated ion channelsthat selectively allow Cl³¹ ions to pass through the plasma membraneupon binding of GABA. GABA_(A) receptors are expressed in neuronsthroughout the central nervous system (CNS) and mediate most of thephysiological activities of GABA in the CNS. Within neurons, the typeand density of GABA_(A) receptors can vary between cell bodies anddendrites. GABA_(A) receptors are also expressed in other tissues,including Leydig cells, placenta, immune cells, liver, bone growthplates, and other endocrine tissues. Outside the CNS, GABA_(A) receptorscan regulate cell proliferation and immune responses.

Structurally, GABA_(A) receptors are pentamers that include fivepolypeptide subunits. The polypeptide subunits are encoded by 19 genesthat are grouped as follows based on sequence similarity: α(1-6),β(1-3), γ(1-3), δ,ε,θ,π, and ρ(1-3). Most subtypes are heteropentamersthat include two copies of one type of α subunit, two copies of one typeof β subunit, and one copy of one type of γ,δ,ε,θ, or π subunit; othersubtypes are homopentamers or heteropentamers of ρ subunits. Knownsubtypes of GABA_(A) receptors include α1β1γ2, α1β2γ2, α1β3γ2, α2β1γ2,α2β2γ2, α2β3γ2, α3β1γ2, α3β2γ2, α3β3γ2, α4β1γ2, α4β3δ, α4β3γ2, α5β1γ2,α5β2γ2, α5β3γ2, α6β1γ2, α6β2γ2, and α6β3γ2. GABA_(A) receptor subtypesvary among tissue types and anatomical regions of the CNS, and subtypesmay be associated with specific functions. In addition, GABA_(A)receptor subtypes may vary between normal and malignant cells of thesame tissue type.

The active site of a GABA_(A) receptor is the binding site for GABA andfor drugs such as muscimol, gaboxadol, and bicuculline. GABA_(A)receptors also have several allosteric binding sites that are thetargets of other drugs, including benzodiazepines, nonbenzodiazepines,neuroactive steroids, barbiturates, ethanol, inhaled anesthetics, andpicrotoxin. Thus, the activity of GABA_(A) receptors is controlled bybinding of molecules to both the active and allosteric binding sites.The structure, function, and regulation of GABA_(A) receptors are knownin the art and described in, for example, Sigel E., and Steinmann, M.E., Structure, Function, and Modulation of GABA_(A) Receptors, J. Biol.Chem. 287:48 pp. 40224-402311 (2012), doi: 10.1074/jbc.R112.386664, thecontents of which are incorporated herein by reference.

The isomerically pure compositions of the invention preferentiallypotentiate the activity selected GABA_(A) receptor subtypes. Thecompositions of the invention may preferentially potentiate the activityof one or more GABA_(A) receptor subtypes, such as those describedabove, relative to one or more GABA_(A) receptor subtypes. In certainembodiments, the compositions preferentially potentiate the activity ofα4β3δ receptors compared to α1β2γ2 receptors.

The compositions of the invention may potentiate one or more GABA_(A)receptors by any mechanism. For example, and without limitation, theisomerically pure form a compound may potentiate a GABA_(A) receptor byallosteric modulation, activation, or inhibition. The allostericmodulation may be positive or negative.

The preferential activity of a composition on one or more GABA_(A)receptor as compared to one or more other GABA_(A) receptor may bemeasured by any suitable means. Activity may be measure using in vitroassays or in vivo assays. For example and without limitation, methods ofmeasuring the effect of modulators on GABA_(A) receptor activity includeanticonvulsant assays, binding assays, fluorescence membrane potentialassays, immune response assays, intracranial self-stimulation assayspatch clamps assays, proliferation assays receptor occupancy assaysseizure induction assays, e.g., using pentylenetetrazol (PTZ) or maximalelectroshock (MES), and survival assays. Such assays are known in theart and described in, for example, International Publication No. WO2016/061527; Ghisdal P., et al., Determining the relative efficacy ofpositive allosteric modulators of the GABA_(A) receptor: design of ascreening approach, J Biomol Screen. 2014 March; 19(3):462-7. doi:10.1177/1087057113501555, Epub 2013 Aug. 29; Tian J., et al., Clinicallyapplicable GABA receptor positive allosteric modulators promote β-cellreplication, Sci Rep. 2017 Mar. 23; 7(1):374. doi:10.1038/s41598-017-00515-y; and Tian J., et al., A Clinically ApplicablePositive Allosteric Modulator of GABA Receptors Promotes Human β-CellReplication and Survival as well as GABA's Ability to InhibitInflammatory T Cells, J Diabetes Res. 2019 Feb. 26; 2019:5783545, doi:10.1155/2019/5783545, the contents of each of which are incorporatedherein by reference.

The preferential activity of a composition on one or more GABA_(A)receptors as compared to one or more other GABA_(A) receptors may beexpressed by any suitable means. For example and without limitation, thepreferential activity may be indicated by a comparison of EC₅₀ values orbinding affinity values.

In certain embodiments, compositions of the invention have an EC₅₀ forα4β3δ GABA_(A) receptors that is lower than the EC₅₀ for α1β2γ2 GABA_(A)receptors. The EC₅₀ for α4β3δ GABA_(A) receptors may be lower than theEC₅₀ for α1β2γ2 GABA_(A) receptors by about 2-fold, about 3-fold, about4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about10-fold, about 20-fold, about 50-fold, about 100-fold, about 200-fold,about 500-fold, or about 1000-fold.

In certain embodiments, compositions of the invention have an EC₅₀ forα4β3δ GABA_(A) receptors that is less than about 50%, less than about40%, less than about 30%, less than about 25%, less than about 20%, lessthan about 15%, less than about 10%, less than about 5%, less than about4%, less than about 3%, less than about 2%, less than about 1%, lessthan about 0.5%, less than about 0.2%, or less than about 0.1% of theEC₅₀ for α1β2γ2 GABA_(A) receptors.

In certain embodiments, compositions of the invention have an bindingaffinity (which may be expressed, e.g., as a dissociation constantK_(D)) for α4β3δ GABA_(A) receptors that is lower than the bindingaffinity for α1β2γ2 GABA_(A) receptors. The binding affinity for α4β3δGABA_(A) receptors may be lower than the binding affinity for α1β2γ2GABA_(A) receptors by about 2-fold, about 3-fold, about 4-fold, about5-fold, about 6-fold, about 7-fold, about 8-fold, about 10-fold, about20-fold, about 50-fold, about 100-fold, about 200-fold, about 500-fold,or about 1000-fold.

In certain embodiments, compositions of the invention have an bindingaffinity for α4β3δ GABA_(A) receptors that is less than about 50%, lessthan about 40%, less than about 30%, less than about 25%, less thanabout 20%, less than about 15%, less than about 10%, less than about 5%,less than about 4%, less than about 3%, less than about 2%, less thanabout 1%, less than about 0.5%, less than about 0.2%, or less than about0.1% of the binding affinity for α1β2γ2 GABA_(A) receptors.

In certain embodiments, compositions of the invention have an EC₅₀ forα4β3δ GABA_(A) receptors that is below a defined value. For example andwithout limitation, the composition may have an EC₅₀ for α4β3δ GABA_(A)receptors that is less than about 1 μM less than about 500 nM, less thanabout 400 nM, less than about 300 nM, less than about 200 nM, less thanabout 100 nM, less than about 50 nM, less than about 25 nM, less thanabout 10 nM, less than about 5 nM, less than about 2.5 nM, less thanabout 1 nM, less than about 0.5 nM, less than about 0.25 nM, or lessthan about 0.1 nM.

In certain embodiments, compositions of the invention have an bindingaffinity for α4β3δ GABA_(A) receptors below a defined value. For exampleand without limitation, the composition may have an binding affinity forα4β3δ GABA_(A) receptors that is less than about 1 μM, less than about500 nM, less than about 400 nM, less than about 300 nM, less than about200 nM, less than about 100 nM, less than about 50 nM, less than about25 nM, less than about 10 nM, less than about 5 nM, less than about 2.5nM, less than about 1 nM, less than about 0.5 nM, less than about 0.25nM, or less than about 0.1 nM.

The compositions and methods of the invention may be effective fortreatment of a GABA_(A) disorder. The GABA_(A) disorder may be anydisease, disorder, or condition associated with altered GABA_(A)receptor function or any disorder may be disease, disorder, or conditionthat can be ameliorated by altered GABA_(A) receptor function. TheGABA_(A) disorder may be acute pain, an addictive disorder, Alzheimer'sdisease, Angelman's syndrome, anti-social personality disorder, ananxiety disorder, attention deficit hyperactivity disorder (ADHD), anattention disorder, an auditory disorder, autism, an autism spectrumdisorder, bipolar disorder, chronic pain, a cognitive disorder, acompulsive disorder, a convulsive disorder, dementia, depression,dysthymia, an epileptic disorder, essential tremor, epileptogenesis,fragile X syndrome, generalized anxiety disorder (GAD), Huntington'sdisease, injury related pain syndrome, insomnia, ischemia, Lewis bodytype dementia, a memory disorder, migraines, a mood disorder, movementdisorder, a neurodegenerative disease, neuropathic pain, an obsessivecompulsive disorder, pain, a panic disorder, Parkinson's disease, apersonality disorder, posttraumatic stress disorder (PTSD), psychosis,Rett syndrome, a schizoaffective disorder, schizophrenia, aschizophrenia spectrum disorder, a seizure disorder, a sleep disorder,social anxiety disorder, status epilepticus, stress, stroke, tinnitus,traumatic brain injury (TBI), vascular disease, vascular malformation,vascular type dementia movement disorder, Wilson's disease, orwithdrawal syndrome.

The methods of treating a subject include providing a composition of theinvention, as described above, to the subject. Providing may includeadministering the composition to the subject. The composition may beadministered by any suitable means, such as orally, intravenously,enterally, parenterally, dermally, buccally, topically (includingtransdermally), by injection, nasally, pulmonarily, and with or on animplantable medical device (e.g., stent or drug-eluting stent or balloonequivalents). Preferably, the composition is provided orally.

The composition may be provided under any suitable dosing regimen. Forexample, the composition may be provided as a single dose or in multipledoses. Multiple doses may be provided in provided separated byintervals, such as 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, ormore. Multiple doses may be provided within a period of time. Forexample, multiple doses may be provided over a period of 1 day, 2 days,3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more. Thecompositions may be provided repeatedly for a specified duration. Forexample and without limitation, the compositions may be provided for 1week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 3 months, 4months, 5 months, 6 months, 8 months, 10 months, 12 months or more.

EXAMPLES Example 1

The ability of CV-10155 and SPNC-019 to modulate the activity ofGABA_(A) receptors of different GABA_(A) was analyzed. CV-10155 andSPNC-019 have the following structures:

Cells expressing the indicated GABA_(A) receptor subtype were exposed togamma-aminobutyric acid in the presence of varying concentrations ofeither CV-10155 or SPNC-019, calcium flux was measured using afluorometric imaging plate reader (FLIPR), and EC50 values for compoundswere determined. Results are provided in Table 1.

TABLE 1 EC₅₀ (M) GABA_(A) Compound CV-10155 SPNC-019 α1β1γ2 5.21E−07 —α1β2γ2 8.39E−07 — α1β3γ2 5.20E−07 — α2β1γ2 2.53E−07 — α2β2γ2 2.13E−07 —α2β3γ2 2.96E−07 — α3β1γ2 9.46E−07 — α3β2γ2 1.82E−06 — α3β3γ2 2.73E−07 —α4β1γ2 2.04E−07 — α4β3δ  1.06E−07 — α4β3γ2 1.33E−06 — α5β1γ2 7.62E−07 —α5β2γ2 3.59E−07 1.198e−006 α5β3γ2 1.30E−06 1.805e−006 α6β1γ2 3.59E−07 —α6β2γ2 1.10E−06 — α6β3γ2 2.45E−07 1.766e−006 — value not measurable

CV-10155 showed some level of positive allosteric modulating activity inall of the GABA_(A) receptor subtypes tested. In contrast, SPNC-019 hadno modulating activity in 15 of the 18 GABA_(A) receptor subtypestested. The only structural difference between CV-10155 and SPNC-019 isthe stereochemical configuration of the hydroxyl and methyl groupsattached to the carbon atom at position 3 of the steroid core. Thus, theresults show that a change in the stereochemistry of a single chiralcenter of a steroid-based compound dramatically alters ability of themolecule to modulate GABA_(A) receptor activity. The results furtherindicate that the isomeric purity of neurosteroid compositions greatlyimpacts the utility of such compositions as therapeutic agents.

Example 2

The ability of various neurosteroids to compete witht-butylbicyclophosphorothionate (TBPS), a ligand for the picrotoxinbinding site of GABA_(A) receptors, was analyzed in InternationalPublication No. WO 2016/061527. WO 2016/061527, pages 215-227. Compoundswere assayed for binding to GABA receptors in membranes isolated fromthe cortices of rat brains. WO 2016/061527, page 216.

Among the neurosteroids analyzed was Compound 10, which has thefollowing structure:

WO 2016/061527, page 106. Compound 10 is identical to the structure ofFormula (II) and is a stereoisomer of the structure of Formula (I).Compound 10/Formula (II) and Formula (I) are stereoisomers that differonly in the configuration of the hydrogen atom bonded to the carbon atomat position 5: Compound 10/Formula (II) has a 5β configuration, whereasFormula (I) has a 5α configuration.

Another neurosteroid analyzed in WO 2016/061527 was Compound 121, whichhas the following structure:

WO 2016/061527, page 150. Compound 121 is a regioisomer of the structureof Formula (I). Compound 121 and Formula (I) differ only in thepositioning of the cyano substituent on the pyrazole ring: Compound 121is substituted at the 3 position of the pyrazole ring, whereas Formula(I) is substituted at the 4 position of the pyrazole ring.

Compound 10 and Compound 121 are isomers that have two structuraldifferences: the stereochemical configuration at carbon 5, and theposition of the cyano substituent on the pyrazole ring.

Results of the analysis are provided in Table 1 of WO 2016/061527. WO2016/061527, pages 217-227. Compound 10 has an IC₅₀ of <10 nM in theTBPS displacement assay, whereas Compound 121 has an IC₅₀ of 10-50 nM.WO 2016/061527, pages 217 and 221.

These results show that subtle structural differences in a neurosteroidcan drastically affect binding of the molecule to GABA_(A) receptors.

Example 3

The pharmacological efficacy of various neurosteroids for α1β2γ2GABA_(A) receptors and α4β3δ GABA_(A) receptors and was analyzed inInternational Publication No. WO 2016/061527. WO 2016/061527, pages227-231. Compounds were tested for the ability to modulate GABA-mediatedcurrents at a submaximal dose of agonist in LTK cells stably transfectedwith α1β2γ2 subunits and in CHO cells transiently transfected with α4β3δsubunits WO 2016/061527, pages 227-228. Cells were incubated with GABAat 2 μM, which is the EC₂₀ for GABA, and 0.01 μM, 0.1 μM, 1 μM, or 10 μMneurosteroid. WO 2016/061527, pages 227-228.

Results of the analysis are provided in Table 2 of WO 2016/061527. WO2016/061527, pages 229-231. Results are presented as the relativepotentiation of GABA-mediated conductance in the presence of 10 μMneurosteroid compared to GABA-mediated conductance in the absence ofneurosteroid. WO 2016/061527, page 228. Compound 121 at 10 μM displayedan efficacy of >500% for both α1β2γ2 GABA_(A) receptors and α4β3δGABA_(A) receptors. WO 2016/061527, page 229.

The results show that a regioisomer of Formula (I) displays nopreferential modulation of α4β3δ GABA_(A) receptors over α1β2γ2 GABA_(A)receptors. In particular, a compound that differs from Formula (I) onlyby the positioning of the cyano substituent on the pyrazole ring hascomparable efficacy on the two GABA_(A) receptor subtypes. Thus, thedata give no indication that compositions containing a compound ofFormula (I) can preferentially modulate α4β3δ GABA_(A) receptors overα1β2γ2 GABA_(A) receptors or that such compositions can be administeredat concentrations that modulate α4β3δ GABA_(A) receptors but not α1β2γ2GABA_(A) receptors. Consequently, nothing from the results suggests thatcompositions containing the compound of Formula (I) would be useful fortreatment of conditions in which potentiation of α4β3δ GABA_(A)receptors but not α1β2γ2 GABA_(A) receptors is beneficial.

In contrast, the data provided in Example 1 show that the compound ofFormula (I) is substantially more active on α4β3δ GABA_(A) receptorsthan on α1β2γ2 GABA_(A) receptors. Taken together, the results in theExamples demonstrate that subtle structural differences in aneurosteroid affect the ability of the molecule to potentiate specificsubtypes of GABA_(A) receptors. Therefore, it follows from the resultsthat the isomeric purity of neurosteroid compositions can influencereceptor subtype specificity and thus the utility of such compositionsas therapeutic agents.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification, and guidance that can be adapted to the practice ofthis invention in its various embodiments and equivalents thereof.

What is claimed is:
 1. A controlled-release oral pharmaceuticalcomposition comprising: an isomerically pure form of a compound ofFormula (I):

and one or more agents that promote controlled release of the compoundof Formula (I) in the gastrointestinal tract of a subject when thecomposition is provided orally to the subject, wherein: the compound ofFormula (I) is present in a therapeutically effective amount topreferentially potentiate an α4β3δ GABA_(A) receptor as compared to anα1β2γ2 GABA_(A) receptor; the isomerically pure form is at least 95%pure by weight with respect to both stereoisomers and regioisomers ofthe compound of Formula (I); the isomerically pure form comprises lessthan 2% by weight of a compound of Formula (II):

and the isomerically pure form comprises less than 2% by weight of acompound of a compound of Formula (III):


2. The composition of claim 1, wherein the compound of Formula (I)preferentially positively modulates an α4β3δ GABA_(A) receptor ascompared to an α1β2γ2 GABA_(A) receptor.
 3. The composition of claim 2,wherein an EC₅₀ of the compound of Formula (I) for an α4β3δ GABA_(A)receptor is less than 50% of an EC₅₀ of the compound of Formula (I) foran α1β2γ2 GABA_(A) receptor.
 4. The composition of claim 3, wherein anEC₅₀ of the compound of Formula (I) for an α4β3δ GABA_(A) receptor isless than 20% of an EC₅₀ of the compound of Formula (I) for an α1β2γ2GABA_(A) receptor.
 5. The composition of claim 1, wherein an EC₅₀ of thecompound of Formula (I) for an α4β3δ GABA_(A) receptor is less than 500nM.
 6. The composition of claim 1, wherein the composition is effectivefor treatment of a GABA_(A) disorder.
 7. The composition of 6, whereinthe GABA_(A) disorder is selected from the group consisting of acutepain, an addictive disorder, Alzheimer's disease, Angelman's syndrome,anti-social personality disorder, an anxiety disorder, attention deficithyperactivity disorder (ADHD), an attention disorder, an auditorydisorder, autism, an autism spectrum disorder, bipolar disorder, chronicpain, a cognitive disorder, a compulsive disorder, a convulsivedisorder, dementia, depression, dysthymia, an epileptic disorder,essential tremor, epileptogenesis, fragile X syndrome, generalizedanxiety disorder (GAD), Huntington's disease, injury related painsyndrome, insomnia, ischemia, Lewis body type dementia, a memorydisorder, migraines, a mood disorder, movement disorder, aneurodegenerative disease, neuropathic pain, an obsessive compulsivedisorder, pain, a panic disorder, Parkinson's disease, a personalitydisorder, posttraumatic stress disorder (PTSD), psychosis, Rettsyndrome, a schizoaffective disorder, schizophrenia, a schizophreniaspectrum disorder, a seizure disorder, a sleep disorder, social anxietydisorder, status epilepticus, stress, stroke, tinnitus, traumatic braininjury (TBI), vascular disease, vascular malformation, vascular typedementia movement disorder, Wilson's disease, and withdrawal syndrome.8. The composition of 7, wherein the GABA_(A) disorder is an epilepticdisorder, epileptogenesis, or a seizure disorder.
 9. The composition ofclaim 1, wherein the composition is formulated for oral administration.10. The composition of claim 1, wherein the composition is formulated asa single daily dosage.
 11. The composition of claim 1, wherein theweight of the compound of Formula (II) makes up less than 1% of thecombined weight of the compound of Formula (I) and the compound ofFormula (II).
 12. The composition of claim 1, wherein the weight of thecompound of Formula (III) makes up less than 1% of the combined weightof the compound of Formula (I) and the compound of Formula (III). 13.The composition of claim 1, wherein the composition comprises a solidformat.
 14. The composition of claim 1, wherein the one or more agentscomprise a solid format.